Process for preparing N6-substituted adenosine derivatives

This is a continuation of International Patent Application No. PCT/US99/29643, filed Dec. 14, 1999, which is, in turn, a continuation of U.S. patent application Ser. No. 60/113,077 filed Dec. 21, 1998, now abandoned.

This invention is directed to a process for preparing N6-substituted adenosine derivatives, to intermediates useful therefor and to methods of preparing these intermediates.

N6-substituted adenosine derivatives, as exemplified by [1S-[1α,2β,3β,4α(S*)]]-4-[7-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl-2,3-dihydroxycyclopentanecarboxamide are useful as cardiovascular agents, more particularly as antihypertensive and anti-ischemic agents, as cardioprotective agents which ameliorate ischemic injury or myocardial infarct size consequent to myocardial ischemia, and as an antilipolytic agents which reduce plasma lipid levels, serum triglyceride levels, and plasma cholesterol levels. See U.S. Pat. Nos. 5,364,862 and 5,561,134.

Methods of preparing these N6 adenosine derivatives and intermediates thereto are disclosed in U.S. Pat. Nos. 5,364,862 and 5,561,134 and International Patent Application Nos. PCT/US97/11320, PCT/US97/15729 and PCT/US97/21439.

This invention is directed to a process for preparing a 2-halo-3-nitro-4-aminopyridine compound of formula

in which a 2,4-dihalo-3-nitropyridine compound of formula

wherein X 1 and X 2 are independently Cl or F, is reacted with an amine of formula H 2 N—X—(Y) a -Z; wherein X is a straight or branched chain alkylene, cycloalkylene or cycloalkenylene group; Y is NR 4 , O or S; a=0 or 1; and Z is of the formula:

where

Z 1 is N, CR 5 , (CH) m -CR 5 or (CH) m -N, m being 1 or 2; Z 2 is N, NR 6 , O or S, n being 0 or 1;

R 4 , R 5 and R 6 are independently H, alkyl, aryl or heterocyclyl; and

R a and R b are independently H, OH, alkyl, hydroxyalkyl, alkyl mercaptyl, thioalkyl, alkoxy, alkoxyalkyl, amino, alkyl amino, carboxyl, acyl, halogen, carbamoyl, alkyl carbamoyl, aryl or heterocyclyl.

In another aspect, this invention is directed to a process for preparing (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine, by reacting a 2,4-dihalo-3-nitropyridine, wherein halo is Cl or F with (R)-1-(3-chlorothien-2-yl)-2-aminobutane, hydrochloride.

The reaction products are process intermediate useful in the preparation of 2,3,4-triaminopyridine compounds. The processes of this invention offers improved yields, purity, ease of preparation and/or isolation of intermediates and final product, and more industrially useful reaction conditions and workability over previously disclosed methods of preparation.

Definitions of Terms

As used above and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

“Acyl” means a straight or branched alkyl-C═O group. “Thioacyl” means a straight or branched alkyl-C═S group. Preferred acyl and thioacyl groups are lower alkanoyl and lower thioalkanoyl having from 1 to about 6 carbon atoms in the alkyl group.

“Alkyl” means a saturated aliphatic hydrocarbon group which may be straight or branched and having about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups may be straight or branched and have about 1 to about 10 carbon atoms in the chain. Branched means that a lower alkyl group such as methyl, ethyl or propyl is attached to a linear alkyl chain.

“Lower alkyl” means an alkyl group having 1 to about 6 carbons.

“Cycloalkyl” means an aliphatic ring having 3 to about 10 carbon atoms in the ring. Preferred cycloalkyl groups have 4 to about 7 carbon atoms in the ring.

“Carbamoyl” means an

group. Alkylcarbamoyl and dialkylcarbamoyl means that the nitrogen of the carbamoyl is substituted by one or two alkyl groups, respectively.

“Carboxyl” means a COOH group.

“Alkoxy” means an alkyl-O group in which “alkyl” is as previously described. Lower alkoxy groups are preferred. Exemplary groups include methoxy, ethoxy, n-propoxy, i-propoxy and n-butoxy.

“Alkoxyalkyl” means an alkyl group, as previously described, substituted by an alkoxy group, as previously described.

“Alkoxycarbonyl means an alkoxy-C═O group.

“Aralkyl” means an alkyl group subsituted by an aryl radical, wherein “aryl” means a phenyl or naphthyl. “Substituted aralkyl” and “substituted aryl” means that the aryl group, or the aryl group of the aralkyl group is substituted with one or more substituents which include alkyl, alkoxy, amino, nitro, carboxy, carbalkoxy, cyano, alkyl amino, halo, hydroxy, hydroxyalkyl, mercaptyl, alkylmercaptyl, trihaloalkyl, carboxyalkyl or carbamoyl.

“Aralkoxycarbonyl” means an aralkyl-O—C═O group.

“Aryloxycarbonyl” means an aryl-O—C═O group.

“Carbalkoxy” means a carboxyl substituent esterified with an alcohol of the formula C n H 2n+1 OH, wherein n is from 1 to about 6.

“Halogen” (or “halo”) means chlorine (chloro), fluorine (fluoro), bromine (bromo) or iodine (iodo).

“Heterocyclyl” means about a 4 to about a 10 membered ring structure in which one or more of the atoms in the ring is an element other than carbon, e.g., N, O or S. Heterocyclyl may be aromatic or non-aromatic, i.e., may be saturated, partially or fully unsaturated. Preferred heterocyclyl groups include pyridyl, pyridazinyl, pyrimidinyl, isoquinolinyl, quinolinyl, quinazolinyl, imidazolyl, pyrrolyll, furanyl, thienyl, thiazolyl, benzothiazolyl, piperidinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, and morpholinyl groups.

“Substituted heterocyclyl” means that the heterocyclyl group is substituted by one or more substituents wherein the substituents include alkoxy, alkylamino, aryl, carbalkoxy, carbamoyl, cyano, halo, heterocyclyl, trihalomethyl, hydroxy, mercaptyl, alkylmercaptyl or nitro.

“Hydroxyalkyl” means an alkyl group substituted by a hydroxy group. Hydroxy lower alkyl groups are preferred. Representative groups include hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl and 3-hydroxypropyl.

Preferred Embodiments

The preparation of N6-substituted adenosine derivatives of formula (I) is outlined in Scheme 1.

In Scheme 1, X 1 and X 2 are independently Cl or F,

Q is CH 2 O;

T is

or R 3 O—CH 2 ;

X is a straight or branched chain alkylene, cycloalkylene or cycloalkenylene group;

Y is NR 4 , O or S;

a=0 or 1;

Z is of the formula

or

Z 1 is N, CR 5 , (CH) m -CR 5 or (CH) m -N, m being 1 or 2;

Z 2 is N, NR 6 , O or S, n being 0 or 1;

R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H, alkyl, aryl or heterocyclyl;

R 7 and R 8 are independently hydrogen, alkyl, aralkyl, carbamoyl, alkyl carbamoyl, dialkyl-carbamoyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aryloxycarbonyl, or R 7 or R 8 together may form

where R c is hydrogen or alkyl,

where R d and R e are independently hydrogen, alkyl, or together with the carbon atom to which they are attached may form a 1,1-cycloalkyl group; and

R a and R b are independently H, OH, alkyl, hydroxyalkyl, alkyl mercaptyl, thioalkyl, alkoxy, alkoxyalkyl, amino, alkyl amino, carboxyl, acyl, halogen, carbamoyl, alkyl carbamoyl, aryl or heterocyclyl.

As shown in Scheme 1, the preparation of N6-substituted adenosine derivatives of formula (I), begins with the reaction of the 2,4-dihalo-3-nitropyridine compound (II) with an amine of formula H 2 N—X—(Y) a -Z (III) provides the 2-halo-3-nitro-4-aminopyridine compound (IV). The reaction is carried out in the presence of a tertiary amine or aromatic amine base such as triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine, 4-methylmorpholine, and the like or an inorganic carbonate base such as sodium carbonate, potassium carbonate, and the like. Preferred bases are 4-methylmorpholine, triethylamine and diisopropylethylamine. The reaction is carried out in a polar aprotic solvent such as 1-methyl-2-pyrrolidone or dimethylformamide, an aromatic solvent such as benzene or toluene, a higher-boiling ethereal solvent such as diglyme or a hindered alcohol such as isopropanol or 2-butanol at a temperature of from about ambient temperature to the reflux temperature of the solvent. A preferred solvent is 1-methyl-2-pyrrolidone, in which the reaction is carried out at about 35° C.

The 2-halo-3-nitro-4-aminopyridine compound (IV) is then reacted with the protected dihydroxyaminocyclopentane compound (V) using the reaction conditions described above to prepare the 2,4-diamino-3-nitropyridine compound (VI).

Reduction of the 2,4-diamino-3-nitropyridine compound (VI) to the 2,3,4-triaminopyridine compound (VII) is accomplished using methods well known in the art for the reduction of aromatic nitro compounds. Preferred methods for the reduction employ metals such as zinc, iron or tin in the presence of an acidic proton source such as ammonium acetate. Solvents are generally alcohols, preferably methanol or mixtures of alcohols and aromatic solvents such as toluene or benzene. The reduction is preferably accomplished at about ambient temperature. The nitro group is also reduced by catalytic hydrogenation, with platinum catalysts such as PtO 2 being preferred.

The groups R 7 and R 8 are then removed using methods well known in the art to form the 2-N-cyclpentane diol-2,3,4-triaminopyridine compound (VIII). In a preferred aspect of this process, R 7 and R 8 together form a dimethylacetonide, in which case the acetonide is hydrolyzed with acid using any solvent which is stable to the acid. A preferred method of acetonide hydrolysis uses HCl and methanol at about ambient temperature. The 2-N-cyclpentane diol-2,3,4-triaminopyridine compound (VIII) may be isolated as the acid solution salt, for example as the dihydrochloride salt, or the acid addition salt may be neutralized with base to form the free amine. In a preferred aspect, the 2-N-cyclpentane diol-2,3,4-triaminopyridine compound (VIII) is isolated as its crystalline acid addition salt. In many instances, the properties of the 2-N-cyclpentane diol-2,3,4-triaminopyridine acid addition salt, including ease of crystallization, are controlled by the particular acid counterion. The counterion in the isolated acid addition salt is selected by treating the free amine with the desired acid, or preferably, by employing an acid having the desired counterion in the acid hydrolysis described above.

The 2-N-cyclpentane diol-2,3,4-triaminopyridine compound (VIII) is then cyclized to the N6-substituted adenosine derivative (I) by reaction with formamidine acetate, formic acid or the ortho esters of formic acid as described in U.S. Pat. Nos. 5,364,862 and 5,561,134 and Intern. Pat. App. No. PCT/US97/21439, incorporated herein by reference. Polar aprotic solvents, higher boiling alcohols and esters and aromatic solvents such as toluene have been employed for these types of cyclizations. Esters such as n-propyl and n-butyl acetate or a combination of one of these esters with ethyl acetate are especially preferred. The N6-substituted adenosine derivative (I) is preferably purified by recrystallization from a suitable organic solvent or mixture of organic solvents. Ethyl, n-propyl and n-butyl acetate and propionate are preferred solvents.

In a preferred aspect of the foregoing process,

Q is CH 2 ;

T is

X is a straight or branched chain alkylene;

a=0;

Z is

Z 1 is N, CR 5 , (CH) m -CR 5 or (CH) m -N, m being 1 or 2;

Z 2 is N, NR 6 , O or S, n being 0 or 1;

R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently H or alkyl;

R 7 and R 8 are independently hydrogen or alkyl, or R 7 and R 8 together may form

where R d and R e are independently hydrogen or alkyl, or together with the carbon atom to which they are attached may form a 1,1-cycloalkyl group; and

R a and R b are independently H, OH, alkyl, hydroxyalkyl, alkyl mercaptyl, thioalkyl, alkoxy, alkoxyalkyl, amino, alkyl amino, carboxyl, acyl, halogen, carbamoyl, alkyl carbamoyl, aryl or heterocyclyl.

In a more preferred aspect, this invention is directed to a process for preparing [1S-[1α,2β,3β,4α(S*)]]-4-[7-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl 2,3-dihydroxycyclopentanecarboxamide as shown in Scheme 2.

As shown in the foregoing Scheme 2, reaction of the 2,4-dihalo-3-nitropyridine compound (II) with (R)-1-(3-chloro-thienyl-2-yl)-2-aminobutane hydrochloride (IX) using the conditions of base and solvent described in Scheme 1 above provides (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine (X) (X 2 is Cl or F). The reaction is conducted in the presence of excess base to neutralize the hydrochloride salt of (IX), or alternatively, the hydrochloride salt of (IX) is neutralized with base separately, and a solution of the free base (IX) is then mixed with a solution of the 2,4-dihalo-3-nitropyridine compound (II).

Preferred 2,4-dihalo-3-nitropyridine compounds are the compounds of formula (II) wherein X 1 is F and X 2 is F or Cl, or a mixture thereof, which are prepared by reaction of 2,4-dichloro-3-nitropyridine with a fluorinating agent such as KF.

The (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine (X) is then reacted with 3aR-[3aα,4α,6a,6aα]-6-amino-N-ethyltetrahydro-3,3-dimethyl-2,4-dioxabicyclo[3.3.0]octan-8-carboxamide benzoate (XI) as described in Scheme 1 above to form [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) which is then reduced as described in Scheme 1 above to form [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII). The reduction is preferably accomplished using zinc, iron or tin in the presence of an acidic proton source such as ammonium acetate, zinc being especially preferred.

Acid hydrolysis of the dimethylacetonide provides [1S-[1α,2β,3β,4α(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentanecarboxamide. The hydrolysis is preferably accomplished using HCl, in which case crystalline [1S-[1α,2β,3β,4α(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentane carboxamide dihydrochloride is isolated directly.

Cyclization of the [1S-[1α,2β,3β,4α(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentanecarboxamide to form [1S-[1α,2β,3β,4α(S*)]]-4-[7-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl 2,3-dihydroxycyclopentane carboxamide (I) is accomplished as described in Scheme 1 above.

In a still more preferred aspect, this invention is directed to a synthesis of [1S-[1α,2β,3β,4α(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentane carboxamide dihydrochloride hydrate effected in a concatenated manner without isolation of the intermediate compounds (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine (X), [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) and [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII).

In particular, 2,4-dihalo-3-nitropyridine is reacted with (R)-1-(3-chloro-thienyl-2-yl)-2-aminobutane hydrochloride in a reaction vessel using the condition of base and solvent described above to form a solution of (R)-N-[1-[(3-chlorothien-2-yl)methyl]propyl]-2-halo-3-nitro-4-pyridinamine (X) in the reaction vessel. 3aR-[3aα,4α,6a,6aα]-6-amino-N-ethyltetrahydro-3,3-dimethyl-2,4-dioxabicyclo [3.3.0]octan-8-carboxamide benzoate (XI) and base are then added to the solution in the vessel and the mixture is heated until conversion to [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-nitropyrid-2-yl-amino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) is essentially complete.

The reaction mixture contained in the vessel is the mixed with water and the aqueous phase, which contains water soluble solvents and impurities, is removed from the vessel.

An alcoholic solvent such as methanol, followed by ammonium acetate and zinc powder, is then added to the organic solution remaining in the vessel and the reaction mixture is heated until reduction of the [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) to [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2yl)methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII) is essentially complete.

The zinc metal and any other insoluble impurities are then removed by filtration of the contents of vessel and the filtrate is collected in another reaction vessel. Acid hydrolysis of [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII) is then effected by adding concentrated HCl to the second reaction vessel and heating until conversion to [1S-[1α,2β,3β,4α(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentanecarboxamide (XIII) is essentially complete. Cooling of the second reaction vessel results in crystallization of [1S-[1α,2β,3β,4α(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentane carboxamide dihydrochloride hydrate which is isolated by filtration.

In another still more preferred aspect, this invention is directed to the concatenated synthesis described above further comprising converting 2,4-dichloro-3-nitropyridine to 2,4-difluoro-3-nitropyridine, or a mixture of 2,4-difluoro-3-nitropyridine and 2-chloro-4-fluoro-3-nitropyridine in the reaction vessel R 1 .

In order to successfully concatenate reaction steps as describe above, solvents and reagents for accomplishing each of the individual reaction steps are selected such that excess reagents and reaction byproducts are readily removed by filtration or extraction, or can remain in the reaction vessel without causing detrimental effects on subsequent reactions performed in the vessel. Accordingly, the following reagents and solvents are preferred.

(1) For conversion of 2,4-dichloro-3-nitropyridine to 2,4-difluoro-3-nitropyridine; KF; tetraphenylphosphonium bromide; 1-methyl-2-pyrrolidinone; toluene.

(1) For conversion of 2,4-dihalo-3-nitropyridine to [3aR-[3aα,4α, 6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-nitropyrid-2-ylamino]N-ethyltetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII): 1-methyl-2-pyrrolidinone; triethylamine; diisopropylethylamine; 4-methyl morpholine.

(2) For conversion of to [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[(1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-nitropyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XII) to [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII): ethyl acetate, methanol, zinc, ammonium acetate.

(3) For conversion of to [3aR-[3aα,4α,6a(R*),6aα]]-6-[4-[[1-[(3-chlorothien-2-yl)methyl]propyl]amino]-3-aminopyrid-2-ylamino] N-ethyl tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxamide (XIII) to [1S-[1α,2β,3β,4α(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate: ethyl acetate, concentrated HCl.

The concatenated synthesis of [1S-[1α,2β,3β,4α(S*)]]-4-[3-amino-4-[[1-[3-chlorothien-2-yl)methyl]propyl]amino]pyrid-2-ylamino] N-ethyl 2,3-dihydroxycyclopentanecarboxamide dihydrochloride hydrate described above, in which the intermediate compounds are not isolated but rather are carried forward to the subsequent reaction in solution, minimizes the four step process to two reaction vessels and two filtrations, resulting in a significant reduction in cycle time from weeks to 1-2 days, elimination of pressurized hydrogenation techniques not easily accommodated or handled in conventional reactions, elimination of the isolation of oily or tar products, crystallization of previously uncrystallizable materials, increased purity of product and significant increase in ease of handling. The concatenated synthesis also significantly impacts the ease of crystallization and increased purity of the final drug substance, [1S-[1α,2β,3β,4α(S*)]]-4-[7-[[1-[(3-chlorothien-2yl)methyl]propyl]amino]-3H-imidazo[4,5-b]pyrid-3-yl] N-ethyl 2,3-dihydroxycyclopentane carboxamide.

The foregoing may be better understood by reference to the following Examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention.